Autism and the measurement of urinary amino acids

Today's post concerns the findings reported by Aiping Liu and colleagues [1] who, following the analysis of urine samples from a group of children diagnosed with an autism spectrum disorder (ASD) and a not-autism control group, concluded that there may be something to see with regards to the urinary excretion of amino acids.

First things first, amino acids are the building blocks of proteins. Long chains of amino acids form different proteins (and peptides) that serve multiple biological functions. But making up proteins is but one of the roles of amino acids, as a variety of other functions are also included in their repertoire; notably also being the raw material for the formation of some neurotransmitters and related compounds (see here for example).

Liu et al approached their analysis of amino acids in relation to autism from the point of view of their measurement being "potential novel metabolic biomarkers for ASD." This follows something of a trend in autism research circles whereby patterns of certain amino acids and their associated chemistry in certain biofluids might have such 'potential' for some types of autism (see here and see here and see here for some other examples) albeit with certain caveats. Researchers utilised some quite well known methods when it came to their analysis - "liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analysis" - and set to work using a tried-and-tested method (see here): "a two-step discovery–validation approach."

Analysing urine samples from nearly 60 children with autism and over 80 not-autism controls ("28 ASD and 41 TD  [typically developing] children for the discovery stage and from an additional cohort of 29 ASD and 41 TD children for the validation stage"), researchers reported detecting and identifying "63 UAA [urinary amino acid] indicators." Twenty-one of these amino acids and/or amino acid metabolites were observed to be "present at significantly different levels in the urine of ASD children compared with TD children" in both participant sets. These compounds were fairly evenly either higher or lower in the kids with autism group (10 higher and 11 lower). I was particularly interested to see that creatinine was observed to be in the significantly higher category associated with the autism group given some other results that were counter to this finding (see here and see here) including some of my own published data [2] from a few years back. Authors also mention how they "identified a panel of 7 UAA indicators that [most effectively] discriminated between the samples from ASD and TD children (lysine, 2-aminoisobutyric acid, 5-hydroxytryptamine, proline, aspartate, arginine/ornithine, and 4-hydroxyproline)."

From those compounds, a few themes emerged with regards to the biochemistry that *might* show some involvement with autism. So: "Abnormalities in the Methionine Cycle in Children With ASD", "Evidence of High Oxidative Stress Levels in Children With ASD" and "Abnormalities in 5HT Metabolism in Children With ASD" are some of the systems potentially implicated by Liu et al. Needless to say that such biological systems are by no means strangers to autism research (see here and see here for examples) albeit not necessarily always in the same direction as the Liu findings.

Caveats? Well yes, a few, such as a reliance solely on single spot urine samples rather than multiple samples from the same person, no other measures of amino acid content in blood for example, and the focus on participants diagnosed with autism excluding things like "attention-deficit hyperactivity disorder, obsessive compulsive disorder" where 'real-life autism' rarely exists in some sort of diagnostic vacuum (see here). But, the findings are interesting and once again highlight how metabolomics is something particularly valuable to autism research (see here) and complementary to genetic studies for example, when trying to decipher the very heterogeneous autisms (plural). Issues with certain amino acids when identified in the context of autism *might* also point to a wider issue (see here) that could also indicate intervention too...

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[1] Liu A. et al. Altered urinary amino acids in children with autism spectrum disorders. Front. Cell. Neurosci. 2019. Jan 10.

[2] Whiteley P. et al. Spot urinary creatinine excretion in pervasive developmental disorders. Pediatr Int. 2006 Jun;48(3):292-7.

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Methyl B12 for autism? Placebo-controlled results say maybe...

"Methyl B12 treatment improved clinician-rated symptoms of ASD [autism spectrum disorder] that were correlated with improvements in measures of methionine metabolism and cellular methylation capacity."

Those were the very encouraging results published by Robert Hendren and colleagues [1] who can now update their ClinicalTrials.gov study entry (see here). Building on the ideas that: "Children with autism spectrum disorder (ASD) have been reported to have reduced ability to methylate DNA and elevated markers of oxidative stress" (topics that have been covered on this blog before), researchers undertook a gold-standard trial - randomised, placebo-controlled - to ascertain the effect (if any) of "8 weeks of treatment with methyl B12 (75 μg/kg) or saline placebo every 3 days in a subcutaneous injection." The success of the treatment was measured by "the Clinical Global Impressions-Improvement (CGI-I) score" accompanied by "changes in the Aberrant Behavior Checklist (ABC) and the Social Responsiveness Scale (SRS)" scores. At the same time, researchers also looked at various biochemical parameters pertinent to "methionine methylation and antioxidant glutathione metabolism."

Based on the 50 children ("mean age 5.3 years") who completed the study, researchers reported a trend of improvement in autistic and related behaviours following the methyl B12 injections. Importantly, the primary outcome measure - the CGI-I scores - rated by clinicians, showed a trend of being "statistically significantly better (lower) in the methyl B12 group (2.4) than in the placebo group (3.1) (0.7 greater improvement in the methyl B12 group, 95% CI 1.2-0.2, p = 0.005)." Biological parameters also showed changes: "increases in plasma methionine (p = 0.05), decreases in S-adenosyl-l-homocysteine (SAH) (p = 0.007) and improvements in the ratio of S-adenosylmethionine (SAM) to SAH (p = 0.007), indicating an improvement in cellular methylation capacity" following the use of methyl B12 compared with placebo.

Accepting that 'subcutaneous injection' of methyl B12 is hardly a 'user-friendly' option and may very well scupper plans to use this particular intervention option for quite a few, these are potentially important results. I'm really quite interested in how vitamin B12 'vitamers' might show some links to at least some 'types' of autism (see here) including the measurement of 'brain levels' of the stuff (see here). The Hendren results suggest that quite a few more research resources might be needed in this area. I wonder also if this future research agenda would include the 'baby and bathwater' compound that is methylmalonic acid in relation to autism too (see here)?

I do also have to point out that previous research from members of this research team has not been so complimentary about the use of methyl B12 in cases of autism [2] despite the idea that there may be 'responders' to this type of intervention. To quote: "methyl B12 may alleviate symptoms of autism in a subgroup of children, possibly by reducing oxidative stress. An increase in glutathione redox status (GSH/GSSG) may provide a biomarker for treatment response to methyl B12." Such differences in reported results are not unfamiliar to autism research (the rule rather than the exception) but perhaps provides a further focus for clarification of effect.

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[1] Hendren RL. et al. Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. J Child Adolesc Psychopharmacol. 2016 Feb 18.

[2] Bertoglio K. et al. Pilot study of the effect of methyl B12 treatment on behavioral and biomarker measures in children with autism. J Altern Complement Med. 2010 May;16(5):555-60.

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Hendren RL, James SJ, Widjaja F, Lawton B, Rosenblatt A, & Bent S (2016). Randomized, Placebo-Controlled Trial of Methyl B12 for Children with Autism. Journal of child and adolescent psychopharmacology PMID: 26889605

Decreased brain levels of vitamin B12 in autism

I have to thank Dr Malav Trivedi for bringing my attention to some recent findings reported by Yiting Zhang and colleagues (including Malav) [1] (open-access) suggesting that: "levels of vitamin B12, especially its MeCbl [methylcobalamin] form, decrease with age in frontal cortex of control human subjects."

Further, researchers reported: "abnormally lower total Cbl [cobalamin] and MeCbl levels in subjects with autism and schizophrenia, as compared to age-matched controls." Some media on the findings can also be read here.

Working from the lab of Dr Richard Deth (quite a familiar name to this blog), researchers initially analysed a most precious sample medium (postmortem brain samples) obtained from various biobanks and including various patient groups. So alongside samples from 12 children with autism were samples from 9 people diagnosed with schizophrenia and some 43 'controls' with ages ranging between 19 weeks old and 80 years old. "Changes in Cbl species were compared with the status of methylation and antioxidant pathway metabolites" accompanied by data derived from a knock-out mouse model: "the influence of decreased GSH [glutathione] production on brain Cbl levels was evaluated in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice in which GSH synthesis was impaired, leading to a brain GSH level decrease of 60–70%."

Looking at postmortem frontal cortex brain samples, researchers reported that finding on levels of vitamin B12 - particularly the MeCbl vitamer -  decreasing with age. Bearing in mind the relatively small participant numbers included, the idea that lower brain tissue levels of total cobalamin and methylcobalamin were also present (almost unanimously) in the autism and schizophrenia groups could be important. I might at this point direct readers to previous discussions on vitamin B12 and autism on this blog (see here) including the research idea of supplementing (see here) with no medical advice given or intended.

There are a few other details worth pointing out from the Zhang findings. Analysis of thiols in brain samples across the autism vs control group revealed some potentially interesting data. So, methionine levels were quite a bit lower in the autism group [significantly lower] as were levels of "the methyl donor S-adenosylmethionine (SAM)." Both these compounds form an important part of the whole 'methylation of DNA' process (see here) among other things.

Glutathione, a compound that has seen its fair share of speculation with autism in mind (see here), was also on the research menu in the Zhang study. Interestingly and again bearing mind the small participant numbers, brain levels of this stuff were lower in the autism group as a whole but not significantly so when compared to controls. This finding might map on to other brain studies with autism in mind (see here). Likewise, cysteine (another potentially relevant compound to some autism) produced a similar finding.

I would encourage readers to take some time looking at the Zhang paper. In conjunction with other results reporting on some important elements to the emerging story (see here) I believe there are further studies to be done applicable to the notion that: "impaired methylation may be a critical pathological component" for at least some autism (see here). Indeed, other research papers have also discussed this issue [2]. The idea that studies about human ageing may likewise be informative to autism (and schizophrenia) research also carries quite a lot of traction too.

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[1] Zhang Y. et al. Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PLoS One. 2016 Jan 22;11(1):e0146797.

[2] Keil KP. & Lein PJ. DNA methylation: a mechanism linking environmental chemical exposures to risk of autism spectrum disorders? Environmental Epigenetics. 2016; 1-15.

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Zhang Y, Hodgson NW, Trivedi MS, Abdolmaleky HM, Fournier M, Cuenod M, Do KQ, & Deth RC (2016). Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia. PloS one, 11 (1) PMID: 26799654

The big H and schizophrenia

Frankly I am more than a little interested in all-things homocysteine when it comes to behaviour and psychiatry. Perhaps more readily finding discussion and argument with regards to more physical health complaints as per the literature on homocysteine and cardiovascular disease risk*, the 'big H' ties into quite a lot of other interesting areas such as folic acid and the link with another important amino acid, methionine and all that methylation mumbo-jumbo.

Islands in the CpG stream @ Wikipedia 

With autism in mind, the various investigations looking at homocysteine have been pretty much all one direction: elevated levels detected in various biological fluids** (open-access). I hate to make generalisations about the autisms, but homocysteine does appear to be something in need of more detailed consideration as per another potential elephant in the room.

Today however my interest turns to a paper by Makoto Kinoshita and colleagues*** (open-access version here) who report that their cohort of participants with schizophrenia (n=42) not only presented with significantly elevated levels of plasma total homocysteine but also that this homocysteine load might also affect DNA methylation.

OK, a recap. Homocysteine and methionine take part in a merry dance together which crosses one-carbon metabolism (folic acid) and should eventually result in methionine being converted to SAM which then donates a methyl group for the process of DNA methylation. I've kinda covered this process on a previous post with autism in mind complete with hand drawn diagram by yours truly.

Kinoshita et al used some nifty technology (quantitative high-resolution DNA methylation array) to look at the methylation status of CpG islands (see here) located across whole gene regions based on the analysis of peripheral leukocytes. They concluded that alongside those elevations in homocysteine "plasma total homocysteine might affect DNA methylation across whole gene regions" and when talking about genes which have been linked to schizophrenia, one might be minded to look at methylation outside of more structural changes to the genome.

Outside of what I've already discussed about the big 'H' and methylation, there is another potentially important implication from all this talk about epigenetics and schizophrenia: intervention. I'm taken back for example to some interesting work about folic acid and SNPs in schizophrenia which was discussed on this blog (see here). The question being whether any intervention strategy designed to bring the homocysteine-methionine relationship back into planetary alignment would impact on gene methylation and then on presented symptoms? (Bearing in mind my caveat about not giving medical advice on this blog).

To finish, I can't talk about (CpG) islands without linking to an interpretation of a famous song about islands.... (what's occurring?)

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* Wald DS. et al. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ. 2002 Nov 23;325(7374):1202.

** Kałużna-Czaplińska J. et al. A focus on homocysteine in autism. Acta Biochim Pol. 2013 Jun 6.

*** Kinoshita M. et al. Plasma total homocysteine is associated with DNA methylation in patients with schizophrenia. Epigenetics. 2013 Apr 26;8(6).

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Kinoshita M, Numata S, Tajima A, Shimodera S, Imoto I, & Ohmori T (2013). Plasma total homocysteine is associated with DNA methylation in patients with schizophrenia. Epigenetics : official journal of the DNA Methylation Society, 8 (6) PMID: 23774737

Methionine synthase and autism

The paper by Christina Muratore and colleagues* (open-access) including Dick Deth and Antonio Persico in the authorship line-up, is the source of today's post. Concerned with quite an important enzyme, methionine synthase (MS), and in particular MS mRNA status in post-mortem frontal cortex samples, the authors report lower levels of MS mRNA in cases of autism. I should add that quite a good overview of this paper can also be found here.

Recycle @ Wikipedia  

OK, let's start from the beginning here. Methionine synthase (MS) is an important enzyme concerned with the regeneration of methionine from homocysteine.

Homocysteine or the 'big H' has been mentioned on more than one occasion on this blog with autism in mind (see here and quite recently here). Indeed, the relationship between methionine and homocysteine intersects a number of other important cycles including those related to folate metabolism and the important methyl-giving properties of SAMe (see this post to see what I'm talking about) and further down the line, that all-important glutathione link (see this post). Oh and it's vitamin B12 dependent.

Anyhow...

• In this study, levels of messenger RNA (mRNA) - an important part of the translation of DNA to proteins - for MS were studied in post-mortem brain samples from deceased person who were diagnosed with autism (n=10) and control, not-autism persons (n=41). Ages at death ranged from 4-30 years for the autism group and 28 weeks - 83 years for the control group.
• Based on the application of qRT-PCR, MS mRNA status across the lifespan of samples included suggested a "striking age-dependent decrease in mRNA levels". In other words, the older the person at time of their death, the less MS mRNA levels were detected in the frontal cortex samples. That being said, they didn't observe corresponding alterations to the level of MS protein despite this age-related change. 
• With the autism group specifically in mind and depending on the primers used to detect specific domains of MS, mRNA levels were reduced compared to controls. The caveat being that again, levels of MS protein were not different when comparing autism vs. controls. That and the suggestion of a lack of an age-dependent decrease in MS mRNA in autism compared to that observed across control samples. 
• Oh and the fact that addition of the pro-inflammatory cytokine TNF-α also seemed to affect levels of MS mRNA.
• There were also some additional findings reported which warrant further attention. So when looking at some of the main players related to that methionine cycle (including methionine, homocysteine, glutathione, etc) in frontal cortex samples (via HPLC) in autism (n=10) and control (n=8) samples, only two parameters came up different: lower, yes lower, homocysteine levels in the autism group alongside lower cystathione levels. Immediately I'm taken back to the recent paper by Jill James discussed quite recently (see here) and their cautions on the use of peripheral markers to denote what might be happening in the brain, albeit with the caveat that the Muratone group was quite a small group.

There are some other details included in this paper regarding "alternative splicing of MS mRNA" but I wouldn't pretend to know all the ins-and-outs of these findings. Suffice to say that there is a suggestion that oxidative stress might have some role to play in what happens to MS both over the course of normal ageing and potentially also in cases of autism.

There's not a great deal more to add about this paper. Yes, again research with a reliance on post-mortem brain samples and all the caveats that go alongside their use (cause of death, comorbidity, etc.). Again however we are presented with some tantalising data about the processes around an important enzyme which has quite a bit of research around it with autism in mind. That alongside some interesting differences found between measures in brain compared with other peripheral tissues which starts to ask some interesting questions about the application of such secondary measures.

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* Muratore CR. et al. Age-dependent decrease and alternative splicing of methionine synthase mRNA in human cerebral cortex and an accelerated decrease in autism. PLoS ONE. 2013; 8: e56927.

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Muratore, C., Hodgson, N., Trivedi, M., Abdolmaleky, H., Persico, A., Lintas, C., De La Monte, S., & Deth, R. (2013). Age-Dependent Decrease and Alternative Splicing of Methionine Synthase mRNA in Human Cerebral Cortex and an Accelerated Decrease in Autism PLoS ONE, 8 (2) DOI: 10.1371/journal.pone.0056927

Pristine cysteine-matically done

First of all, sorry for the terrible pun that makes up the post title. What can I say apart from (a) what else rhymes with the amino acid cysteine? and (b) I'd probably make a terrible tabloid newspaper headline maker unlikely to come up with something like this British classic.

In this post I want to focus on a paper by Mostafa Waly and colleagues* (full-text) which includes a couple of notable names on the authorship list including Dick Deth (macroepigenetics and high-fructose corn syrup) and Mady Hornig (carbohydrate digestion and the bacteria which just rolls of the tongue, Sutterella in relation to autism).

The name of the paper's game is cysteine uptake in autism, and how issues with this process may have some interesting connections to "inadequate antioxidant capacity" and onwards affecting prenatal- and postnatal epigenetic programming. I have to admit that this paper does jump around quite a bit in terms of what might impact on what so don't be surprised if I start bringing quite disparate areas into this post. I'll also say now that ultimately this is a paper of mouse models and how an old friend, autoimmuunity, might play some role in cysteine uptake. I'll stress the 'might play some role' before I progress any further.

A few descriptions first:

The amino acid cysteine has cropped up previously on this blog. Not only linked to those very important observations on sulphate (sulfate) levels in various biofluids in cases of autism but also with regards to the growing interest in glutathione (GSH) and autism as a result of cysteine being one of the building blocks of GSH and the various brain revelations published not so long ago.

Epigenetics... well, you could have a look at this post from a few months back introducing epigenetics in relation to autism. The mantra: your genome might not necessarily be your destiny just about covers the science of epigenetics and potentially how epigenetics might resolve some of the issues in the grudge match that is genes vs. environment. I've posted about this elsewhere quite recently (here).

Anyway back to the Waly paper. It is open-access but here are a few of the highlights:

• Unless I am missing something, it is not immediately clear whether this is a summary paper, an experimental-type paper or some combination of the two. After a few reads, I favour the latter option because aside from introducing the important processes involved in cysteine metabolism and epigenetics, there does appear to be some practical experimentation on various types of cell and tissue derived from animal models; in particular the C57BL/6  and SJL/J mouse models. Unfortunately no room for the BTBR Dangermouse model of autism.
• Indeed the practical experiment side of things seemed to involve a few things including: (a) extracting things like regulatory (CD4+ CD25+) T-cells from the mouse models to ascertain the expression of EAAT3, a mediator of cysteine uptake in various body sites (see here) (b) analysis of the level of GSH in the frontal cortex of said mouse models treated with or without the mercury-based preservative thiomersal (or thimerosal) which has been the focus of quite a lot of discussion over the years, and (c) analysis of the activity of methionine synthase, the enzyme that converts homocysteine to methionine, again in the cortex of thiomersal treated and untreated mice.
• A few of the results, but don't quote me on this: GSH levels in the frontal cortex of the SJL/J mice were lower than the C57BL/6 mice. This might make a little more sense if I point you towards some evidence that the SJL/J mouse has been described as quite a good model of autoimmunity, or at least slightly better than the C57BL/6 model.
• Similarly, levels of methionine synthase activity were described as lower in the SJL/J mice.
• It appears that thiomersal treatment had very little effect on GSH or methionine synthase activity results.
• The EAAT3 results, remembering that EAAT3 transports cysteine, cysteine from dietary sources, into cells partly for GSH synthesis. "Expression of EAAT3 was significantly lower in CD4+ T-cells from SJL/J mice versus C57BL6/J mice, suggesting that autoimmunity is associated with impaired capacity for cysteine uptake."

I'll admit that I have scratched my head a few times when reading this paper. The title suggests epigenetic programming to be a core part of the presented evidence but ultimately the data seems to focus more on the speculation around the mouse model differences over any experimental data on a specific epigenetic role tied to autism.

Don't get me wrong, the GSH and methionine synthase expression findings are important and I would love to see how they might compare against the BTBR mouse model of autism bearing in mind its representativeness to autism (see this paper by Pobbe and colleagues** full-text). The additional fact that thiomersal treatment seemed to have very little effect on these parameters in both mouse models is also a potentially important finding.

That being said I almost got the impression that this paper might have been better split into two manuscripts: one on the speculated mechanisms, which provide an excellent overview it has to be said, and another on the fact that C57BL/6 mice don't tend to show as many issues with cysteine, glutathione and methionine pathways as the SJL/J mouse. I caution though that this last finding might not necessarily translate into real life autism.

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* Waly M. et al. Prenatal and postnatal epigenetic programming: implications for GI, immune, and neuronal function in autism. Autism Research & Treatment. 2012
DOI: 10.1155/2012/190930

** Pobbe RL. et al. Expression of social behaviors of C57BL/6J versus BTBR inbred mouse strains in the visible burrow system. Behavioral Brain Research. 2010; 214: 443-449.